1. Field of the Invention
The present invention relates to a method of analyzing autoradiograph for determining the base sequence of nucleic acids.
2. Description of the Prior Art
It is essential to obtain genetic information carried by organisms in order to make the function or replication mechanism of the organism clear in the field of molecular biology and biotechnology which have been rapidly developed in recent years. Particularly, it is essential to determine the base sequence of nucleic acids such as DNA (or DNA fragment; the same applies hereinbelow) which carries specific genetic information.
Maxam-Gilbert method and Sanger-Coulson method are known as typical methods for determining the base sequence of nucleic acids such as DNA and RNA. In the former Maxam-Gilbert method, a group containing a radioactive isotope such as .sup.32 P is attached to a chain molecule of DNA at one end to label it with the radioactive element and the bond between the constitutional units of the chain molecule is base-specifically cleaved by a chemical reaction. A mixture of the resulting base-specific DNA cleavage products is resolved (developed) through gel electrophoresis to obtain a resolved pattern (not visible) wherein each of the numerous cleavage products is resolved on the gel support medium. The resolved pattern is visualized on a radiographic film such as an X-ray film to obtain an autoradiograph thereof as a visible image. The bases in the certain positional relationships with the end of the radioactive element-attached chain molecule can be sequentially determined according to the visualized autoradiograph and the applied base-specific cleavage means. In this way, the sequence for all bases of the DNA specimen can be determined.
In the latter Sanger-Coulson method, synthetic DNA products which are complementary to the chain molecule of DNA and radioactively labeled, are base-specifically synthesized by utilizing a chemical reaction, and the obtained mixture of numerous synthetic DNA products is resolved on a support medium by gel electrophoresis to obtain a resolved pattern. In a similar manner to that described above, the base sequence of DNA can be determined according to the visualized autoradiograph.
The base sequence of the nucleic acids has been conventionally determined by visually judging individual resolved positions (bands) of the base-specific cleavage products or the base-specific synthetic products of radioactively labeled nucleic acid (hereinafter referred to simply as base-specific fragments of nucleic acid) on the visualized autoradiograph and comparing the positions of the bands among them. Namely, the analysis of the autoradiograph is done by observing the visualized autoradiograph with eyes, and such visual analysis requires great amounts of time and labor. Further, since the visual analysis of the autoradiograph varies or fluctuates owing to the skill of investigators, the results on the determination of the base sequence of nucleic acid vary depending on the investigators and the accuracy of information is limited to a certain extent.
For instance, the analysis of the autoradiograph is made in such a manner that an X-ray film having the visualized autoradiograph is fixed onto a sharcastene and then a cursor made of a plastic (a measure plate for reading) placed on the sharcastene is manually moved on the film up and down (or right and left) to visually judge the relative positions of the radioactively labeled substances (bands) using the cursor as a measure. On the center of the transparent plastic cursor (so-called hair line cursor), a fine straight line is usually drawn. The hair line cursor can be moved in one direction (in the direction parallel to the film), while fixed to the sharcastene. When the radioactively labeled substances are ideally resolved, that is, when resolved rows are approximately straight and the radioactively labeled substances of each row are resolved at the same velocity, the relative positions of bands among the resolved rows can be easily determined by using the straight cursor to thereby sequence the bands.
However, the resolved pattern often causes a smiling phenomenon or offset distortion. The smiling phenomenon is a phenomenon in which migration distances of the radioactively labeled substances at the both sides of the support medium are shorter than that in the vicinity of the center thereof. This phenomenon is caused by heat dissipation effect (so-called edge effect), etc. during the resolving procedure such as electrophoresis. The offset distortion is a phenomenon in which the positions of resolved rows (lanes) are wholly deviated from one another and is caused by difference between the slots in the resolution-starting positions or time of samples, which is due to the unevenness of the shapes of slots, etc. Besides the deviation of the bands from one another, there is a possility that the disorder of the bands themselves and the zigzag of the lanes are caused.
In such a case, the positional relationship between the bands cannot be judged by using the hair line cursor alone, because the cursor is straight, fixed to the device and allowed only to make a parallel movement. Analyzers have paid close attention to the determination of the sequence of bands and corrected the deviation of positions and the disorder with eyes so as not to misread the sequence of bands. Accordingly, the hair line cursor is only a rough measure for reading.
Alternatively, an analyzing method utilizing a computer system using a digitizer board, etc. has been attempted, in which the X-ray film having the visualized autoradiograph is fixed onto the digitizer board which has two-dimensional coordinates and is online to the computer system and then names of bases to which bands are assigned are inputted through other inputting means such as a keyboard while indicating the bands on the film with an indicating stick. According to this method, sequence and base name are automatically given to each band based on the coordinates inputted by the indicating stick.
There is another problem that the read bands and unread bands are hardly distinguished from each other in the above-mentioned methods using the sharcastene and the computer system. These methods have further disadvantages in that eyes in reading the bands on the X-ray film differs from eyes upon the keyboard to input base name and that there is a time interval therebetween. Thus, the reversal of the sequence of bands, misreading such as overlapping or overlooking of bands, or the mis-inputting of base names cannot be prevented.
The obtained base sequence of nucleic acids has been recorded and stored in a recording material such as a paper or in a recording medium such as a magnetic disk. Since the information on the base sequence determined on the basis of the autoradiograph are stored in a medium different from that on which the information on the autoradiograph of the resolved pattern is recorded, namely the X-ray film, it is difficult that both are allowed to collate to each other.
More in detail, the relationship between the resolved pattern and base sequence information included in the pattern is clear during the analysis of the autoradiograph. However, after the analysis is completed and base sequence information is recorded on other medium, it is very difficult to make one of them correspond to the other again. Hence, there are further problems that the analysis must be done all over again when the analysis is interrupted, or it requires much time and labor same as in the analysis when the analytical results are verified. Pattern information and base sequence information are respectively valuable as a series of information, both infomation being recorded and stored in the different mediums in the different forms. However, it is impossible that the both mediums are allowed to correspond one-to-one to each other immediately.
There have been disclosed by the present applicant (or the assignee) in U.S. patent application Nos. 664,405, abandoned and continued in U.S. Ser. Nos. 07/423,686 and 837,037, abandoned and continued in U.S. Ser. No. 07/378,509, autoradiographic procedures which utilize a radiation image recording and reproducing method using a stimulable phosphor sheet for obtaining the autoradiograph of radioactively labeled substances resolved on a support medium, in place of the conventional radiography using a radiosensitive film. Said stimulable phosphor sheet comprises a stimulable phosphor and has such properties that when exposed to a radiation, the stimulable phosphor absorbs a portion or radiation energy and then emits light (stimulated emission) corresponding to the radiation energy stored therein upon excitation with an electromagnetic wave (stimulating rays) such as visible light or infrared rays. According to this method, exposure time can be greatly shortened and there is no fear of causing problems such as chemical fog associated with prior arts. Further, since the autoradiograph having information on radioactively labeled substances is stored in the phosphor sheet as radiation energy and then read out photoelectrically as stimulated emission, the autoradiograph can be directly obtained as digital signals and then recorded on a suitable recording medium to store it.
There are also proposed in U.S. patent application Nos. 568,875, now U.S. Pat. No. 4,868,746, No. 568,877, now abandoned, and continued as U.S. patent application No. 07/024,909, now U.S. Pat. No. 4,777,597, No. 849,187, abandoned and continued as U.S. patent application No. 07/541,197 and No. 854,381, now U.S. Pat. No. 4,720,786, methods for automatically determining the base sequence of nucleic acids by obtaining the autoradiograph as digital signals and subjecting the digital signals to appropriate signal processing.